Plasmids are routinely used in the preparation of recombinant proteins and in the preparation of DNA for gene therapy purposes. The stable maintenance of a plasmid in a host cell is important for the efficient preparation of these products. However, extrachromosomal DNA carried in host cells is inherently unstable due to an increased metabolic burden on cells containing the plasmid compared to cells that are plasmid-free. To maintain plasmid stability and decrease metabolic burden, plasmids have been engineered to contain dominant selectable markers.
The conventional method of maintaining plasmids in cells in culture is to include an antibiotic resistance gene on the plasmid and to culture the cells in the presence of the appropriate antibiotic. For cells or plasmids intended for therapeutic use, this has the disadvantage that use of plasmid containing the antibiotic resistance gene may contribute to the spread of antibiotic resistance.
Some methods of plasmid maintenance have attempted to exploit naturally-occurring post segregational killing mechanisms controlled by plasmid-borne genes. For example, the hok/sok, srnB and pnd systems involve a killer protein encoded by a stable mRNA and regulated by a small, unstable antisense RNA that binds to the killer RNA and inactivates it. The killer RNA is retained in plasmid-free segregants after the antisense RNA has degraded and is translated into the lethal protein. Plasmid maintenance using the hok/sok system was investigated in the attenuated live vector vaccine strain Salmonella typhi CDV 908 htr-A (Galen et al, 1999, Infect. Immunol, 67: 6424-6433). However, such post-segregational killing mechanisms do not enable plasmid selection following transformation and are therefore still dependent on the presence of an antibiotic resistance gene on the plasmid.
Some alternative methods for maintaining and selecting plasmids without antibiotic selection have been developed in which the plasmid encodes a gene complementing a host cell auxotrophy. For example, a host cell may be a mutant cell which is unable to synthesise an essential amino acid metabolite and which can only survive in medium lacking the amino acid in the presence of a plasmid comprising a gene encoding the missing element for synthesis of this amino acid (Wang M-D et al, 1987, J. Bacteriol., 169: 5610-5614). However, this approach limits the composition of the growth medium since the amino acid must be omitted. An alternative method, which can be used in complex media, uses a mutant host cell with a thermosensitive tRNA synthetase gene which can only survive at non-permissive temperatures if a plasmid comprising the wild-type tRNA synthetase gene is present (Skogman et al, 1984, Gene 31: 117-122). Another selection method uses a plasmid-borne tRNA gene to complement nonsense mutations in essential chromosomal genes in a mutant host cell (Zengel et al, 1981, J. Bacteriol, 145: 459-465). Alternatively, a gene that increases the metabolic burden on a cell, such as the pil operon, may be placed on the host chromosome such that the host cell only survives in the presence of a plasmid encoding the corresponding repressor protein (Ogden et al, 1992, Biotech. Bioeng., 40:1027-1038).
EP 0851932 describes a method of maintaining plasmids within host cells in in vitro culture by means of operator repressor titration. The method involves engineering a host cell, such that it contains a first chromosomal gene encoding a repressor and a second chromosomal gene essential for cell growth that has an operator sequence for the repressor in its control region. In the absence of a plasmid, expression of the second chromosomal gene is inhibited by binding of the repressor to the operator and the cell dies. The plasmids for maintenance in this host cell are engineered to contain the operator sequence such that in the presence of the plasmid, the repressor is titrated away from the operator for the gene essential for cell growth, the gene is expressed and the cell survives. This mechanism is also described in Williams et al (Nucleic Acids Research, 1999, 26(9): 2120-2124) and in Cranenburgh et al (Nucleic Acid Research, 2001, 29(5): e26-e27).
Although some mechanisms of plasmid maintenance and selection which do not rely on antibiotic selection are known, there remains a need for the development of additional methods in view of the increasing importance of plasmids in the production of DNA and recombinant proteins for therapeutic applications. In addition, the systems of plasmid maintenance and selection developed to date require the use of plasmids which have been specially modified for use in these systems. There remains a need for a system of plasmid maintenance and selection which does not involve antibiotic resistance and which employs plasmids that are common in the art and do not require special modification.